Original Research published: 27 November 2017 doi: 10.3389/fimmu.2017.01669
Anti-idiotypic Antibodies against BP-IgG Prevent Type XVII Collagen Depletion Mayumi Kamaguchi 1,2, Hiroaki Iwata1*, Yuiko Mori 1, Ellen Toyonaga1, Hideyuki Ujiie1, Yoshimasa Kitagawa 2 and Hiroshi Shimizu 1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 2 Department of Oral Diagnosis and Medicine, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan
Edited by: Falk Nimmerjahn, University of Erlangen-Nuremberg, Germany Reviewed by: Bruce David Mazer, The Research Institute of the McGill University Health Center, Canada Jan Lunemann, University of Zurich, Switzerland *Correspondence: Hiroaki Iwata
[email protected] Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology Received: 29 August 2017 Accepted: 14 November 2017 Published: 27 November 2017 Citation: Kamaguchi M, Iwata H, Mori Y, Toyonaga E, Ujiie H, Kitagawa Y and Shimizu H (2017) Anti-idiotypic Antibodies against BP-IgG Prevent Type XVII Collagen Depletion. Front. Immunol. 8:1669. doi: 10.3389/fimmu.2017.01669
Bullous pemphigoid (BP) mainly targets type XVII collagen (COL17). Intravenous immunoglobulin (IVIg) is used to treat numerous autoimmune diseases, including BP. The major mechanism of action for IVIG is thought to be its immunomodulatory effect. However, little is known about the precise mechanisms of IVIg in BP. We investigate the cellular effects of IVIg, toward treatments for BP. Keratinocytes were treated with IgG from BP patients (BP-IgG) and with IVIg, and then the COL17 expression was detected by Western blotting. Cell adhesion and ex vivo dermal–epidermal separation were also investigated for the condition with BP-IgG and IVIg. BP-IgG targeting the non-collagenous 16A domain induces the depletion of COL17 in cultured keratinocytes (DJM-1 cells). The COL17 levels in DJM-1 cells were decreased by 50% after 4 h of BP-IgG stimulation as determined by Western blotting. By contrast, BP-IgG with IVIg was found to result in 70–90% increases in COL17 and to restore adhesion to the plate. Interestingly, IVIg significantly inhibited the binding of BP-IgG to the COL17-enzymelinked immunosorbent assay plate, and this was due to anti-idiotypic antibodies against BP-IgG. When anti-idiotypic antibodies against BP-IgG in 0.02% of IVIg were depleted from IVIg, those antibodies did not exhibit inhibitory effects on COL17 depletion. When cryosections of human skin were incubated with BP-IgG in the presence of leukocytes, dermal–epidermal separation was observed. BP-IgG treatment with IVIg or anti-idiotypic antibodies did not induce such separation. These findings strongly suggest the presence of anti-idiotypic antibodies against anti-COL17 IgG in IVIg. This mechanism of IVIg could be a target for therapies against BP. Keywords: bullous pemphigoid, type XVII collagen, intravenous immunoglobulin, idiotypic antibody, depletion, autoantibody
INTRODUCTION The first uses of intravenous immunoglobulin (IVIg) were in immunodeficient individuals and individuals with severe infections. IVIg is currently used to treat numerous autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and autoimmune blistering diseases (AIBDs) (1). Controlled studies of IVIg as a treatment for pemphigus and pemphigoid patients found IVIg to be a safe, effective treatment (2, 3). Furthermore, several case reports have Abbreviations: IVIg, intravenous immunoglobulin; AIBD, autoimmune blistering disease; EBA, epidermolysis bullosa acquisita; BP, bullous pemphigoid; COL17, type XVII collagen; NC16A, the non-collagenous 16A domain; RT, room temperature; BSA, bovine serum albumin; FcRn, neonatal Fc receptor; ELISA, enzyme-linked immunosorbent assay.
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described the use of IVIg to treat AIBDs such as pemphigoid and epidermolysis bullosa acquisita (EBA) (4–6). Although various modes of action for IVIg have been proposed in AIBDs, the mechanisms behind its effect are still not fully understood (7, 8). The major mechanism of action for IVIg in AIBDs is thought to be its immunomodulatory effect (1, 9–11). In addition, anti-idiotypic antibodies against pathogenic antibodies have been reported in autoimmune disorders (1, 12). Although there are many autoimmune disorders, anti-idiotypic antibodies against autoantibodies have been proved in only several autoimmune disorders (13–18). Bullous pemphigoid (BP) is the most common AIBD (19). Two autoantigens, type XVII collagen (COL17, also called BP180) and BP230, which form a hemidesmosome, are targeted in BP, and COL17 is particularly relevant to the pathogenesis (19). Antibody-induced tissue damage is a major pathology in autoantibody-mediated autoimmune diseases (20). The activation of complements and/or inflammatory cells, including neutrophils and eosinophils, is crucial to the development of clinical phenotypes in animal models (21–23). In addition, molecular or cellular mechanisms have been proposed. IgG from BP patients (BP-IgG) targeting the non-collagenous 16A (NC16A) domain of COL17 induces the depletion of COL17 in cultured keratinocytes (24). It is thought that the shortage of COL17 causes an insufficiency of hemidesmosomes during remodeling that eventually results in weak cell adhesion to the basement membrane (25). Regarding treatments for BP patients, it has been reported recently that IVIg provides therapeutic benefits to BP patients (3). A randomized, double-blind, placebo-controlled clinical study concluded that IVIg has therapeutic benefits for patients with BP who are resistant to systemic steroid therapy. The inhibition of autoantibody production and inflammatory cascades are major strategies in BP treatment. Prednisolone is thought to have dual effects and is commonly used. In most BP treatments, the targets are immune cells, including neutrophils and antigenspecific B cells and/or T cells. However, little is known about the effects of IVIg in keratinocytes expressing the autoantigens. This study focused on the cellular effects of IVIg, for the treatment of BP.
local guidelines. The studies were conducted in accordance with the Helsinki guidelines.
Anti-COL17 NC16A IgG Purification
Anti-COL17 NC16A IgG was purified from total IgG using a protein G affinity column by means of the HiTrap HNS-activated HP column according to the manufacturer’s instructions (GE Healthcare). Briefly, GST fusion COL17 NC16A was produced as previously described (26). The recombinant protein was coupled with the HiTrap NHS-activated HP column. The titer of anti-COL17-specific IgG was measured by indirect immunofluorescent staining. Indirect immunofluorescent staining using anti-COL17-specific IgG (concentration 0.1 mg/ml) demonstrated titers greater than 1:32,000.
Treatment Agents
Two different IVIgs (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan, and Baxter International Inc., Deerfield, IL, USA; diluted with PBS) were purchased and used for this study. The concentrations of each agent for the treatment are given in Section “Results” and the figure legends.
Depletion of Anti-idiotypic Antibodies
To deplete anti-idiotypic antibodies against anti-COL17 NC16A IgG, anti-COL17 NC16A IgG was coupled to a HiTrap NHSactivated HP column according to the manufacturer’s instructions (GE Healthcare). IVIg was passed through the column to remove anti-idiotypic antibodies, and then flow-through fractions (the “IVIg-depleted” sample) and the elution fraction (the “idiotype” sample) were used for the depletion assay. To evaluate the depletion efficacy, 96-well microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with purified anti-COL17 NC16A IgG (500 ng/well), normal human IgG and PBS. Nonspecific binding was reduced by blocking with protein-free blocking buffer (Thermo Fisher Scientific, Rockford, IL, USA) at room temperature (RT) for 1 h. Plates were subsequently incubated with biotin-conjugated IVIg (1 mg/ml). IVIg was conjugated with biotin using a biotin labeling kit according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). Finally, plates were incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific) for IgG subclasses at RT for 0.5 h.
MATERIALS AND METHODS BP Patients and Total IgG Purification
Cell Culture
The BP patients fulfilled both inclusion criteria: (i) clinical blistering or erosions on the skin and (ii) circulating autoantibodies against COL17 as detected by BP180-NC16A enzyme-linked immunosorbent assay (ELISA)/CLEIA (MBL, Nagoya, Japan). BP-IgG was purified from plasma obtained by apheresis in a severe BP patient. Total IgG was purified using a protein G affinity column according to the manufacturer’s instructions (GE Healthcare, Amersham, UK). In accordance with the Hokkaido University Hospital bylaws and standard operating procedures approved by the Hokkaido University Hospital Review Board, we obtained patient consent for experimental procedures to be performed at Hokkaido University Hospital. A full review and approval by an ethics committee were not required, according to
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DJM-1 cells isolated from human skin squamous cell carcinoma (27) were cultured in DMEM. To investigate the depletion of COL17, cells were cultured to approximately 40% confluence (24). DJM-1 cells were pretreated with agents for 1 h, and then BP-IgG (concentration 1 mg/ml) was added to the culture media for 4-h incubation. In some experiments, DJM-1 cells were treated with BP-IgG for 4 h followed by IVIg for 1 h.
Western Blotting
For Western blot analysis of whole-cell lysates, cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma Aldrich), and the lysates were
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RESULTS
centrifuged. Each fraction was subjected to SDS–PAGE in 6% polyacrylamide gel. The gels were transferred onto nitrocellulose membranes. Blotting was performed using rabbit anti-COL17 (1:2,000 dilution) (26), rabbit anti-β-tubulin (Abcam, Tokyo, Japan, 1:20,000 dilution), and anti-integrin α6 (Santa Cruz, Dallas, TX, USA, 1:500 dilution) as the primary antibodies, followed by incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Life Technologies, 1:5,000 dilution). Signals were visualized with Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA).
IVIg Prevents COL17 Depletion of Keratinocytes Induced by BP-IgG
When DJM-1 cells are treated with BP-IgG, the amount of COL17 is decreased as determined by Western blotting (COL17-depletion assay) (24). In this study, we examined the effects of IVIg by means of a COL17-depletion assay. For a COL17-depletion assay, 40% confluent cells were incubated with BP-IgG (concentration: 1 mg/ml) for 4 h. The amount of COL17 relative to β-tubulin was determined by Western blotting. DJM-1 cells were treated with BP-IgG before (the pretreatment sample) or after (the posttreatment sample) the addition of serially diluted IVIg. BP-IgG without IVIg induced an approximately 50–60% reduction of COL17 in DJM-1 cells. By contrast, BP-IgG pretreated with 5 mg/ml IVIg restored the amount of COL17 by 70–90% (p
